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There are numerous current limitations in phosphoproteomic analysis. Large-scale phosphorylation analysis, in particular the quantitative measurement of changes in phosphorylation, is vital to understanding how signaling networks interact and function, and how they are misregulated in disease states. For the past decade, gel-free shotgun sequencing has become the major strategy for in-depth phosphoproteomic analyses. At present, large-scale phosphoproteome experiments frequently require extensive fractionation, phosphopeptide enrichment of each fraction, long LC gradient for individual mass spectrometric analysis, and integration of large datasets. Although thousands or even tens of thousands of protein phosphorylation sites can be identified, many researchers may be only interested in a limited number of phosphoproteins that show response to a signaling event. Therefore, large-scale phosphoproteomics has become time-consuming and cost-prohibitive for a typical project. On the other hand, gel-based proteomic strategy, which often combines protein separation by 1- or 2-dimensional polyacrylamide gel electrophoresis (1D or 2D-PAGE) with protein identification by mass spectrometry (MS), has pioneered the field of proteomics and has remained a workhorse in proteomic studies. Difference Gel Electrophoresis (DIGE) overcomes the poor reproducibility among gel experiments and is widely used to profile whole proteome expression under different conditions. DIGE allows researchers to visualize proteins expressed differentially before choosing relevant ones for identification by mass spectrometry, typically by high speed MALDI-TOF/TOF. The applicability of gel-based proteomic strategy in phosphoproteomics has been largely limited by the lack of technologies for specific and quantitative detection of phosphoproteins in gels. It was proposed to use 32P/33P radioactive labeling for difference phosphoproteomes. However, it is a serious safety concern, needs large amount of radioactive material to label endogenous proteins, and is not compatible with mass spectrometric analyses. There have been several attempts to stain phosphoproteins for large-scale phosphoproteomes in 2D gels, including commercial products such as Pro-Q Diamond and Phos-tag. While these assays show some promise in terms of versatility, they are limited by low specificity of binding between the incorporated metal ions and phosphate groups. Highly abundant non-phosphorylated proteins can also be stained in these studies, making these methods less reliable for large-scale phosphoproteomes. These existing commercial reagents are therefore typically used for staining or mobility assays of simple samples and cannot be utilized for simultaneous detection and quantitation within the same gel. There is therefore an unmet need for a routine and effective analysis of relevant phosphoproteins.